In the pharmaceutical and biopharmaceutical industry, new therapeutic proteins and existing FDA-approved proteins are often modified with compounds that enhance their physical properties, such as solubility, hydrolytic stability and aggregation, as well as their biomedical properties, such as antigenicity, proteolytic stability, serum circulation time, and ease of delivery. At present, modification with poly(ethylene glycol) (PEG), commonly known as pegylation, is the most widely used modification for therapeutic applications. However, other compounds, such as PEG derivatives and neutral hydrophilic polymers, e.g. dextran, are also useful to this end. The same kind of modification is also applied to other molecules than proteins, such as low molecular weight organic drugs and drug candidates.
PEG-modified proteins and low molecular weight drugs is an important class of biopharmaceuticals, which is commonly produced by pegylation of pre-purified molecules. Once the PEG has been contacted with the pre-purified solution under the appropriate conditions, the reaction mixture so obtained will contain unreacted PEG, unmodified molecules and pegylated molecules. Consequently, a subsequent purification step will be required, wherein the target such as a monopegylated or polypegylated molecules is isolated from the other components of the mixture. Since unreacted PEG exhibits both colloidal and detergent properties, and under some solution conditions may precipitate or cause precipitation of proteins there is a well-known risk of interference in the subsequent purification. For example, if chromatography is used to purify the target, the unreacted PEG could promote fouling of the separation matrix. Accordingly, it is important to be able to efficiently remove unreacted PEG from a process as early as possible.
Ultrafiltration has been suggested to remove unreacted PEG. However, this requires a significant size difference between the PEG and the pegylated molecules, which is not always the case. In addition, ultrafiltration is difficult and costly to scale up, and hence not suitable for large-scale processing.
Chromatography is a well known method for purification of liquids, such as reaction mixtures. In chromatography, two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample. In liquid chromatography, a liquid sample, optionally combined with a suitable buffer constitutes the mobile phase, which is contacted with a stationary phase, known as a separation matrix. Usually, the matrix comprises a support to which ligands, which are groups capable of interaction with the target, have been coupled. The principles of chromatography are well known, and extensively described in the literature. In addition, a large number of patent documents describe development in supports and ligand technology.
For example, U.S. Pat. No. 3,793,299 (Zimmerer, R. E.), which was granted in 1974, discloses an early ion exchange material based on a cellulose support. This patent provides a solution to problems caused by the cellulose's affinity for water. More specifically, it had been recognised that prior art ion exchange materials based on cellulose were difficult to use by consequence of the cellulosic material's tendency to swell, gelatinize or disperse on contact with an aqueous solution. To avoid these problems, U.S. Pat. No. 3,793,299 presents a cation exchange material prepared by grafting onto cellulose a polymerisable vinyl monomer which is either carboxylated or carboxylatable on hydrolysis; and thereafter contacting the grafted cellulose with caustic alcoholic or aqueous solution for about 10-30 minutes; after which the caustic treatment is quenched. The product obtained is a cation exchanger, i.e. when used in chromatography, it will interact with a positively charged target via ionic interactions.
More recently, ion-exchange chromatography was disclosed for purification of pegylated viruses. More specifically, WO 98/39467 (Calydon Inc.) describes purification of pegylated adenovirus using the anion exchanger Q SEPHAROSE™ XL (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Q SEPHAROSE™ XL is a strong anion exchanger comprised of highly crosslinked agarose to which dextran is attached, and its functional groups are quaternary amino.
Another group of ion-exchangers is known as tentacle gels, due to the large tentacle-like groups that extend from the support. One available product marketed for separation of neutral and basic proteins and peptides is FRACTOGEL® EMD COO- (Merck), a weakly acidic cation exchanger wherein the functional groups are carboxyl groups and the solid support is comprised of methacrylate-based copolymer. The functional carboxyl groups are bonded via polyelectrolyte chains enabling the ionic groups to adopt a configuration that is optimal for their electrostatic interaction with the target. Thus, the separation of proteins on FRACTOGEL® EMD COO- is based on reversible electrostatic interactions between the positively charged regions of the protein surface and the support. The strength of the binding depends on the buffer system, the pH value of the buffer which determines the surface charge of the protein as well as the degree of the ionisation of the functional groups of the exchanger, the concentration of the counter ions and the charge density on the support. Elution from FRACTOGEL® EMD COO- is achieved either by high salt concentrations or by decrease of pH.
Finally, it is known that poly(acrylic acid) forms complexes with polyethylene (PEG) in aqueous solutions. Journal of Polymer Science (Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 32, 1385-1387 (1994)) reports that hydrophobic interactions may be of great importance for the stabilisation of such complexes. This is evidenced by the higher stability of a poly(methacrylic acid)-PEG complex than that of the poly(acrylic acid)-PEG complex, which higher stability is explained by the presence of CH3 groups in the poly(methacrylic acid). Further, it appears that subtle changes in acid group structure and underlying matrix may play an important role in this context.